Higher spatial resolution is demanded in the field of observation and measurement of a microstructure in a sample (specimen). As a method of enhancing the spatial resolution, a technique to image a sample with performing modulation using striped illumination light, and performing demodulation the image by image processing has been proposed (e.g. Patent Documents 1 and 2).
The sixth embodiment disclosed in Patent Document 1 is an example applied to a fluorescent observation device, and the optical system thereof splits illumination light, including coherent light, emitted from a light source using a beam splitting element, such as a diffraction grating, then collects the plurality of split illumination light beams at a pupil position of an object lens, and allows the beams to emit as parallel beams having different angles from the objective lens, so as to form interference fringes overlapping in a neighborhood of the observation object. By the illumination light modulated in stripes, diffracted light, including spatial frequency components of the shape formation of the observation object, which cannot be transmitted by the imaging optical system alone, can be used for imaging. Then by relatively modulating the phase of the split illumination light, interference fringes are moved on the observation object and a plurality of images is acquired, so as to form an image by image processing.
In concrete terms, phase modulation to move the interference fringes is performed by moving the beam splitting element in a direction perpendicular to the optical axis, or, according to another example, by inserting a wedge prism in one optical path of the illumination light and moving the wedge prism in a direction perpendicular to the optical axis.
According to the method disclosed in Patent Document 2, illumination light including coherent light emitted from a light source is introduced via an optical fiber, and is then split by a beam splitting unit, such as a diffraction grating, and the plurality of split illumination light is collected at a pupil position of an objective lens, so as to form interference fringes in a neighborhood of the observation object. Just like the above mentioned case, by the illumination light modulated in stripes, high frequency components of the shape information of the observation object, which cannot be transmitted by the imaging optical system alone, can be used for imaging. Further, just like the above mentioned case, a plurality of images is acquired so as to form an image by image processing.
In this method, in order to form one image, not only is a plurality of images acquired with modulating the phase of the illumination light, but also the images are acquired with changing the orientation of the interference fringes of the illumination light. This is because the high frequency components can be used for forming an image only when the structure has the same direction as the direction of the interference fringes of the illumination light, therefore in order to reproduce the shape of the specimen that extends two-dimensionally, it is necessary to acquire and synthesize a plurality of images with changing the direction of the interference fringes.
In order to allow two or more beams to interfere in such a structured illumination, it is normally preferable that the beams are S-polarized light with respect to the interference plane. This is because the contrast when S-polarized light beams are interfered is 1, regardless the incident angles, but contrast when P-polarized light beams enter attenuates in proportion to cos (Δθ) with respect to a difference Δθ of the incident angles of the beams. The value of the contrast of the P-polarized light becomes negative when Δθ>90°, which means that the contrast of the interference fringes is inverted.
Since a structured illumination microscope is a technique used for obtaining high resolution, it is desirable that the numerical aperture (NA) of the objective lens to be used is as large as possible and the fringe cycle of the structured illumination is as short as possible. As a result, the beam for the structured illumination enters the specimen at a large angle, therefore if there is a P-polarized light component, attenuation of the P-polarized light is large because the above mentioned Δθ is large, which deteriorates the structured illumination contrast.